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Indole-3-carbinol (I3C) is a phytochemical that exhibits growth-inhibitory activity against various cancer cells. However, there are limited studies on the effects of I3C on colon cancer cells. In this study, the growth-inhibitory activity of I3C against the human colorectal carcinoma cell line (LoVo) was examined. The results of the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide, colony formation, and cell counting assays revealed that I3C suppressed the proliferation of LoVo cells. Microscopy and wound-healing analyses revealed that I3C affected the morphology and inhibited the migration of LoVo cells, respectively. I3C induced apoptosis and DNA fragmentation as evidenced by the results of fluorescein isothiocyanate-conjugated annexin V staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling assay, respectively. Additionally, I3C arrested the cell cycle at the G0/G1 phase and enhanced the reactive oxygen species levels. Western blotting analysis revealed that treatment with I3C resulted in the activation of apoptotic proteins, such as poly(ADP-ribose) polymerase, caspase-3, caspase-7, caspase-9, Bax, Bim, and p53 in LoVo cells. These results indicate that I3C induces apoptosis in LoVo cells by upregulating p53, leading to the activation of Bax and caspases. Taken together, I3C exerts cytotoxic effects on LoVo cells by activating apoptosis.

MG132 is a potent, reversible, and cell-permeable 20S proteasome inhibitor and it is derived from a Chinese medicinal plant. The purpose of this study is to investigate the anticancer effects of MG132 against human osteosarcoma U2OS cells. We first performed MTT and colony formation assays to investigate the anti-proliferative effects of MG132. The results demonstrated that MG132 suppressed the proliferation of U2OS cells. Furthermore, we found that treatment with MG132 increased apoptosis and induced DNA damage in U2OS cells. Additionally, zymography, wound healing, and invasion assays showed that MG132 suppressed the enzymatic activity of matrix metalloproteinases, cell migration, and invasion, respectively of U2OS cells. Furthermore, western blotting assay was performed to investigate the apoptotic signaling pathways in MG132-treated U2OS cells. Our results showed that MG132 downregulated the expression of antiapoptotic proteins, including CDK2, CDK4, Bcl-xL, and Bcl-2, whereas it upregulated the expression of proapoptotic proteins, including p21, p27, p53, p-p53 (ser15, ser20, and ser46), cleaved forms of caspase-3, caspase-7, caspase-9, and PARP, and FOXO3 in U2OS cells. These results demonstrated that MG132 activated apoptotic signaling pathways in U2OS cells. Interestingly, MG132 downregulated the phosphorylation of Akt and Erk. Taken together, our results suggest that MG132 has anticancer effects in U2OS cells. Therefore, MG132 may be a potential therapeutic agent for the treatment of osteosarcoma.

Benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon formed during the incomplete combustion of organic matter, has harmful effects. Therefore, much research is ongoing to develop agents that can mitigate the effects of B[a]P. The aim of this study was to examine the effect of maclurin, one component of the branches of Morus alba L., on the B[a]P-induced effects in HaCaT cells, a human keratinocyte cell line. Maclurin treatment inhibited aryl hydrocarbon receptor (AHR) signaling as evidenced by reduced xenobiotic response element (XRE) reporter activity, decreased expression of cytochrome P450 1A1 (CYP1A1), and reduced nuclear translocation of AHR. The B[a]P-induced dissociation of AHR from AHR-interacting protein (AIP) was suppressed by maclurin. Maclurin also inhibited the production of intracellular reactive oxygen species (ROS) induced by B[a]P. In addition, the antioxidant property of maclurin itself was demonstrated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. Furthermore, maclurin activated antioxidant response element (ARE) signaling through enhancement of ARE luciferase reporter activity and the expression of ARE-dependent genes including nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and heme oxygenase-1 (HO-1). Nrf2 activation and its nuclear translocation were promoted by maclurin through p38 MAPK activation. These data indicate that maclurin had antagonistic activity against B[a]P effects through activation of Nrf2-mediated signaling and inhibition of AHR signaling and, suggesting its potential in protecting from harmful B[a]P-containing pollutants.

BackgroundCK2α/CSNK2A1 is involved in cancer progression by phosphorylating various signaling molecules. Considering the role of CSNK2A1 in cancer progression and phosphorylation of SIRT6 and the role of SIRT6 in chemoresistance through the DNA damage repair pathway, CSNK2A1 and SIRT6 might be involved in resistance to the conventional anti-cancer therapies.MethodsWe evaluated the expression of CSNK2A1 in the 37 osteosarcoma patients and investigated the effects of CSNK2A1 and phosphorylation of SIRT6 on Ser338 on the resistance to the anti-cancer effects of doxorubicin. Results Higher expression of CSNK2A1 predicted shorter overall survival and relapse-free survival in both general osteosarcoma patients and sub-population of patients who received postoperative chemotherapies. U2OS and KHOS/NP osteosarcoma cells with induced overexpression of CSNK2A1 were resistant to cytotoxic effects of doxorubicin, and knock-down of CSNK2A1 potentiated the cytotoxic effects of doxorubicin. CSNK2A1 overexpression-mediated resistance to doxorubicin was associated with SIRT6 phosphorylation and induction of the DNA damage repair pathway molecules ATM and Chk2. CSNK2A1 and SIRT6 mediated resistance to doxorubicin in vivo was attenuated via mutation of SIRT6 at the Ser338 phosphorylation site. Emodin, a CSNK2A1 inhibitor, potentiated the cytotoxic effects of doxorubicin in osteosarcoma cells in vitro. ConclusionsThis study demonstrates that the expression of CSNK2A1 might be used as a prognostic indicator of osteosarcoma. In addition, it suggests that CSNK2A1 induces resistance to doxorubicin through phosphorylation of SIRT6-mediated activation of the DNA damage repair pathway. Therefore, blocking the CSNK2A1-SIRT6-DNA damage repair pathway might be a new therapeutic stratagem for the poor prognostic subgroup of osteosarcomas with high expression of CSNK2A1.

Background IL4Rα and IL13Rα1 are constituents of the type II IL4 receptor. Recently, IL4Rα and IL13Rα1 were reported to have roles in cancer progression and suggested as potential prognostic markers. However, studies on IL4Rα and IL13Rα1 in soft-tissue sarcomas have been limited. Methods This study investigated the immunohistochemical expression of IL4Rα and IL13Rα1 in 89 soft-tissue sarcomas of the extremities, superficial trunk, and retroperitoneum. Immunohistochemical staining for IL4Rα and IL13Rα1 were scored according to a combination of staining intensity and staining area in tissue microarray samples. Positivity for the immunohistochemical expression of IL4Rα and IL13Rα1 were determined using receiver operating curve analysis. Statistical analysis was performed using regression analysis and a chi-square test. Results In human soft-tissue sarcomas, immunohistochemical expression of IL4Rα was significantly associated with IL13Rα1 expression. Nuclear and cytoplasmic expression of IL4Rα and IL13Rα1 were significantly associated with shorter survival of soft-tissue sarcoma patients in univariate analysis. Multivariate analysis indicated that nuclear expression of IL4Rα and IL13Rα1 were independent indicators of shorter overall survival (IL4Rα; p = 0.002, IL13Rα1; p = 0.016) and relapse-free survival (IL4Rα; p = 0.022, IL13Rα1; p p p Conclusions This study suggests IL4Rα and IL13Rα1 are associated with the progression of soft-tissue sarcoma, and the expression of IL4Rα and IL13Rα1 might be novel prognostic indicators of soft-tissue sarcoma patients.

This study was focused on investigating the anticancer potential of indole-3-carbinol (I3C) against lung cancer H1299 cells via an increase in ROS levels. To investigate the induction of growth arrest and/or cell death in H1299 cells, a cell cycle arrest assay, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL) assay, and reactive oxygen species (ROS) detection assay were performed. Through the TUNEL assay, we detected I3C-induced DNA fragmentation. Fluorescence-activated cell sorting (FACS) analysis showed that I3C induced an increase in ROS levels and apoptotic rate in a dose- and time-dependent manner in H1299 cells. Western blotting demonstrated that activated forms of caspase-3, caspase-7, caspase-9, and poly (ADP-ribose) polymerase (PARP) were increased in I3C-treated H1299 cells following treatment with I3C. Furthermore, protein expression levels of FOXO3, bim, bax, and phosphorylated ERK and JNK were increased, while those of pAkt, Bcl-xL, and Bcl-2 were decreased by I3C treatment of H1299 cells. To confirm the relationship between cell apoptosis and ROS generation, H1299 cells were treated with I3C simultaneously with N-acetylcysteine (NAC), and it was shown that ROS levels decreased and viability increased. Moreover, in western blot analysis, expression of anti-apoptotic proteins (thioredoxin1, peroxiredoxin-1, Bcl-2, and Bcl-xL) in I3C-treated cells was evidently downregulated and pro-apoptotic proteins (active ASK1 and cleaved PARP) were upregulated compared to cells co-treated with NAC. The study showed that I3C induced downregulation of ROS regulator proteins and elevation of ROS, thus activating apoptotic signaling cascades in human lung cancer H1299 cells.

Tomentosin, a sesquiterpene lactone, is known to possess various biological activities. However, its anticarcinogenic activity against human hepatocellular carcinoma (HCC) cells has not been investigated in detail. Thus, this study aimed to elucidate the cytotoxic mechanism of tomentosin in human HCC cell lines HepG2 and Huh7. WST-1, cell counting, and colony formation assay results showed that treatment with tomentosin decreased the viability and suppressed the proliferation rate of HepG2 and Huh7 cells in a dose- and time-dependent manner. Cell cycle analysis revealed increased population of cells at the SubG1 and G2/M stage, and decreased population of cells at the G0/1 stage in HepG2 and Huh7 cells treated with tomentosin. Annexin V/propidium iodide double staining and TUNEL assay results showed increased apoptotic cell population and DNA fragmentation in HepG2 and Huh7 cells treated with tomentosin. Western blotting analysis results showed that tomentosin treatment significantly increased the expression level of Bax, Bim (short form), cleaved PARP1, FOXO3, p53, pSer15p53, pSer20p53, pSer46p53, p21, and p27, but decreased the expression of Bcl2, caspase3, caspase7, caspase9, cyclin-dependent kinase 2 (CDK2), CDK4, CDK6, cyclinB1, cyclinD1, cyclinD2, cyclinD3, and cyclinE in a dose-dependent manner. Taken together, this study revealed that tomentosin, which acted through cell cycle arrest and apoptosis, may be a useful therapeutic option against HCC.

Background Recently, FAM83H was reported to have roles in cancer progression in conjunction with oncogenic molecules such as MYC and b-catenin. Moreover, the data from the public database indicates a molecular relationship between FAM83H and zinc finger proteins, especially between FAM83H and ZNF16. However, studies on FAM83H and ZNF16 in gallbladder cancer have been limited. Methods This study investigated the expression of FAM83H and ZNF16 in 105 gallbladder carcinomas. Results In human gallbladder carcinomas, immunohistochemical expression of FAM83H was significantly associated with ZNF16 expression. In univariate analysis, nuclear and cytoplasmic expression of FAM83H or ZNF16 were significantly associated with shorter survival of gallbladder carcinoma patients. Multivariate analysis revealed the nuclear expression of FAM83H as an independent indicator of poor prognosis of overall survival (p = 0.005) and relapse-free survival (p = 0.005) of gallbladder carcinoma patients. Moreover, co-expression patterns of nuclear FAM83H and ZNF16 were also independent indicators of shorter survival of gallbladder carcinoma patients (overall survival; p p Conclusions This study suggests FAM83H and ZNF16 are associated with the progression of gallbladder carcinoma, and the expressions of FAM83H and ZNF16 might be novel prognostic indicators of gallbladder carcinoma patients.

Since the cause of hypopigmentary skin disorders (hair graying and vitiligo) is typically unknown, there is no known cure for these disorders. Melia azedarach L. is used in Southeast Asia across China and Japan as a traditional medicine, and it has been reported to have various pharmacological properties. However, there have been no reports to demonstrate the involvement of M. azedarach L. in pigmentation. This study was conducted to investigate the effect of ethanolic extract of M. azedarach L. (MAE) on melanogenesis and to elucidate its mechanism of action in B16F10 mouse melanoma cells and human epidermal melanocytes. Effects of MAE on melanogenesis and its mechanism of action were investigated using several assays, including melanin content, cellular tyrosinase activity, real-time PCR analysis, Western blot analysis, and ELISAs for cyclic AMP (cAMP), protein kinase A (PKA), cAMP response element binding (CREB) protein, and mitogen-activated protein kinases (MAPKs). MAE increased the melanin content levels and cellular tyrosinase activity in B16F10 mouse melanoma cells and human epidermal melanocytes. In addition, the action mechanism of MAE-induced melanogenesis was examined in human epidermal melanocytes. It also upregulated the expressions of microphthalmia-associated transcription factor (MITF) gene and its downstream target genes, tyrosinase and tyrosinase-related protein (TRP) 1, but not TRP 2. MAE treatment increased the cAMP levels, PKA activity, and phosphorylation of CREB protein, its downstream signaling protein. However, MAE showed no effects on MAPKs (p42/44 MAPK, p38 MAPK, and c-Jun-N-terminal kinase (JNK)). These findings indicate that MAE induces melanogenesis by upregulating the MITF gene through the cAMP-PKA-CREB signaling pathway, and they suggest its potential in the treatment of hypopigmentary skin diseases.

Many cruciferous vegetables, including cabbage, contain indole-3-carbinol (I3C), which is a known anticarcinogen. However, the anticarcinogenic effects of I3C on liver cancer have not been investigated. Therefore, this study was conducted to evaluate the anticarcinogenic effects of I3C in human hepatocellular carcinoma (HCC) SNU449 cells. The results of MTT and WST-1 assays indicated that treatment of SNU449 cells with I3C decreased viability in dose- and time-dependent manners, while colony formation assays indicated that I3C also inhibited proliferation of SNU449 cells. Moreover, fluorescence-activated cell sorter analysis showed that I3C induced apoptosis in SNU449 cells in dose- and time-dependent manners. Furthermore, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling revealed that I3C induced DNA fragmentation in SNU449 cells in a time-dependent manner, while Western blotting showed that apoptotic proteins such as p53, cleaved PARP, caspase-3, and caspase-7 were activated in SNU449 cells following treatment with I3C. Finally, reactive oxygen species-related protein peroxiredoxin-1 and thioredoxin-1 expression decreased in I3C-treated SNU449 cells. The aim of our study is to investigate the unknown mechanisms responsible for the apoptotic effects of I3C on human HCC SNU449 cells, and the results suggest that I3C may be useful for the prevention and treatment of liver cancer.

In this study, we investigated the inhibitory mechanisms of resorcinol in B16F10 mouse melanoma cells. We found that resorcinol reduced both the melanin content and tyrosinase activity in these cells. In addition, resorcinol suppressed the expression of melanogenic gene microphthalmia-associated transcriptional factor (MITF) and its downstream target genes tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2. In addition, we found that resorcinol reduced intracellular cAMP levels and protein kinase A (PKA) activity, and increased phosphorylation of the p38 mitogen-activated protein kinase (MAPK). Resorcinol was also found to directly inhibit tyrosinase activity. However, resorcinol-induced decrease in melanin content, tyrosinase activity, and tyrosinase protein levels were attenuated by SB203580, a p38 MAPK inhibitor. Taken together, these data indicate that anti-melanogenic activity of resorcinol is be mediated through the inhibition of cAMP signaling and activation of p38 MAPK, indicating that resorcinol may be a possible ameliorating agent in the treatment of hyperpigmentation skin disorders.

Background Broussonetia papyrifera (L.) Ventenat, a traditional medicinal herb, has been applied as a folk medicine to treat various diseases. Broussochalcone A (BCA), a chalcone compound isolated from the cortex of Broussonetia papyrifera (L.) Ventenat, exhibits several biological activities including potent anti-oxidant, antiplatelet, and cytotoxic effects. Purpose The purpose of this study is to elucidate the inhibitory effect of BCA against CYP2J2 enzyme which is predominantly expressed in human tumor tissues and carcinoma cell lines. Study design The inhibitory effect of BCA on the activities of CYP2J2-mediated metabolism were investigated using human liver microsomes (HLMs), and its anti-cancer effect against human hepatoma HepG2 cells was also evaluated. Methods Two representative CYP2J2-specific probe substrates, astemizole and ebastine, were incubated in HLMs with BCA. After incubation, the samples were analyzed using liquid chromatography-tandem mass spectrometry. To investigate the binding model between BCA and CYP2J2, we carried out structure-based docking simulations by using software and scripts written in-house. Results BCA inhibited CYP2J2-mediated astemizole O-demethylation and ebastine hydroxylase activities in a concentration dependent manner with Ki values of 2.3 and 3.7 µM, respectively. It also showed cytotoxic effects against human hepatoma HepG2 cells in a dose-dependent manner with activation of apoptosis related proteins. Conclusion Overall, this was the first report of the inhibitory effects of BCA on CYP2J2 in HLMs. The present data suggest that BCA is a potential candidate for further evaluation for its CYP2J2 targeting anti-cancer activities.

The expression of ANO1 is considered to have diagnostic specificity for gastrointestinal stromal tumors. However, its function as a calcium-activated chloride channel suggests that the expression of ANO1 is not restricted to gastrointestinal stromal tumors. Recently, it has been reported that ANO1 has roles in the progression of human malignant tumors. However, the role of ANO1 in breast carcinoma has been controversial. Therefore, we investigated the expression of ANO1 in 139 breast carcinoma patients and the role of ANO1 in vitro. The immunohistochemical expression of ANO1 was significantly associated with the expression of β-catenin, cyclin D1, MMP9, snail, and E-cadherin. Especially, ANO1 expression was an independent indicator of poor prognosis of shorter overall survival and relapse-free survival of breast carcinoma patients by multivariate analysis. In MCF7 and MDA-MB-231 breast carcinoma cells, inhibition of ANO1 with T16Ainh-A01 or siRNA for ANO1 significantly suppressed the proliferation of cells. Knock-down of ANO1 with siRNA induced G0/G1 cell cycle arrest and significantly inhibited the invasiveness of breast carcinoma cells. Knock-down of ANO1 decreased the expression of β-catenin, cyclin D1, MMP9, snail, and N-cadherin, and increased the expression of E-cadherin. In conclusion, this study demonstrates that ANO1 expression is an indicator of poor prognosis of breast carcinoma patients and suggests that ANO1 might be a therapeutic target for breast carcinoma patients with ANO1-positive tumors and poor prognosis.

Vitiligo and hair graying are acquired chronic hypopigmentary disorders of the skin. This study was carried out to investigate the effect of ethanolic extract of D. odorifera (DOE) on melanogenesis and determine its mechanism of action in B16F10 mouse melanoma cells and human epidermal melanocytes. DOE increased melanin content levels. It also enhanced the expression of microphthalmia-associated transcription factor (MITF) gene and its downstream target genes tyrosinase-related protein (TRP) 1, TRP 2, and tyrosinase. DOE increased cAMP levels, as determined by a cAMP production assay. In addition, DOE increased PKA activity and induced phosphorylation of cAMP response element binding (CRE B) protein, its downstream signaling protein. However, DOE showed no effects on mitogen-activated kinases (MAPKs) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Taken together, DOE induces melanogenesis by upregulating MITF gene through the cAMPCREB signaling pathway, which suggests its potential for the treatment of hypopigmentary skin diseases.

Orostachys japonicus is an herb that contains several functional components and has traditionally been used to treat various diseases in Asia. In this study, bioactive components from different parts of the O. japonicus plant were investigated, and the contents of the functional components in ethanol extracts of O. japonicus cultivated in Korea and China were compared. The antioxidant effects of O. japonicus ethanol extracts were investigated using Raw 264.7 cells. It was found that 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity was significantly decreased in the cells treated with the extracts. Moreover, the novel inhibitory functions of O. japonicus extracts on collagenase, elastase, and tyrosinase were established. We also found that O. japonicus extracts strongly inhibited melanin synthesis in B16F10 melanoma cells by decreasing MITF protein levels and activating the Erk and Akt signaling pathways. Thus, these findings would be useful for developing new cosmetic and pharmaceutical formulations based on O. japonicus extracts.

Melanogenesis plays a critical role in the protection of skin against external stresses such as ultraviolet irradiation and oxidative stressors. This study was aimed to investigate the effects of cannabidiol on melanogenesis and its mechanisms of action in human epidermal melanocytes. We found that cannabidiol increased both melanin content and tyrosinase activity. The mRNA levels of microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein (TRP) 1, and TRP2 were increased following cannabidiol treatment. Likewise, cannabidiol increased the protein levels of MITF, TRP 1, TRP 2, and tyrosinase. Mechanistically, we found that cannabidiol regulated melanogenesis by upregulating MITF through phosphorylation of p38 mitogen-activated protein kinase (MAPK) and p42/44 MAPK, independent of cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling. In addition, the melanogenic effect of cannabidiol was found to be mediated by cannabinoid CB1 receptor, not by CB2 receptor. Taken together, these findings indicate that cannabidiol-induced melanogenesis is cannabinoid CB1 receptor-dependent, and cannabidiol induces melanogenesis through increasing MITF gene expression which is mediated by activation of p38 MAPK and p42/44 MAPK. Our results suggest that cannabidiol might be useful as a protective agent against external stresses.

Nowadays, transplantation of human mesenchymal stem cells (MSCs) has emerged as a potential cellular therapy for liver cirrhosis. Hepatocyte growth factor (HGF) plays an important role in the regeneration of the liver. The objective of the study was to investigate the therapeutic effect of HGF-transfected human umbilical cord blood-derived MSCs on dimethylnitrosamine (DMN)-induced liver fibrosis in rats. HGF-transfected MSCs were transplanted into rats with DMN-induced liver fibrosis. H2O2-induced cytotoxicity, apoptosis and intracellular reactive oxygen species were reduced in HGF-transfected MSCs in HGF-transfected MSCs. Pro-apoptotic proteins, such as cleaved poly (ADP-ribose) polymerase and cleaved caspase-3, were decreased in HGF-transfected MSCs. Biochemical analysis showed that the levels of aspartate aminotransferase and alanine aminotransferase were decreased after transplantation of HGF-transfected MSCs in rat fibrosis. Trichrome staining showed that HGF-transfected MSCs reduced liver damage. Taken together, our study indicated that HGF-transfected MSCs have therapeutic effects on DMN-induced liver fibrosis in rats.

Background FAM83H was initially identified as a protein essential for dental enamel formation. Recent reports have shown that FAM83H is also involved in the progression of human cancers in conjunction with tumor-associated molecules, such as MYC and β-catenin. However, the role of FAM83H in sarcoma has not yet been investigated. Methods The expression and roles of FAM83H and β-catenin were evaluated in human osteosarcomas from 34 patients and osteosarcoma cells. Results The expression of nuclear FAM83H, cytoplasmic FAM83H, and β-catenin were significantly associated with each other and significantly associated with shorter survival of osteosarcoma patients by univariate analysis. In multivariate analysis, cytoplasmic expression of FAM83H was an independent indicator of shorter survival of osteosarcoma patients (overall survival; P

SCRIB is a polarity protein important in maintaining cell junctions. However, recent reports have raised the possibility that SCRIB might have a role in human cancers. Thus, this study evaluated the roles of SCRIB in ovarian cancers. In 102 human ovarian carcinomas, nuclear expression of SCRIB predicted shorter survival of ovarian carcinoma patients, especially in the patients who received post-operative chemotherapy. In SKOV3 and SNU119 ovarian cancer cells, overexpression of SCRIB stimulated the proliferation and invasion of cells. Knockout of SCRIB inhibited in vivo tumor growth of SKOV3 cells and overexpression of SCRIB promoted tumor growth. Overexpression of SCRIB stimulated epithelial-to-mesenchymal transition by increasing the expression of N-cadherin, snail, TGF-β1, and smad2/3, and decreasing the expression of E-cadherin, the converse was observed with inhibition of SCRIB. In conclusion, this study presents the nuclear expression of SCRIB as a prognostic marker of ovarian carcinomas and suggests that SCRIB is involved in the progression of ovarian carcinomas by stimulating proliferation and epithelial-to-mesenchymal transition-related invasiveness.

Background Acetylshikonin is one of the biologically active compounds derived from the root of Lithospermum erythrorhizon, a medicinal plant with anti-cancer and anti-inflammation activity. Although there have been a few previous reports demonstrating that acetylshikonin exerts anti-cancer activity in vitro and in vivo, it is still not clear what is the exact molecular target protein of acetylshikonin in cancer cells. Purpose The purpose of this study is to evaluate the inhibitory effect of acetylshikonin against CYP2J2 enzyme which is predominantly expressed in human tumor tissues and carcinoma cell lines. Study design The inhibitory effect of acetylshikonin on the activities of CYP2J2-mediated metabolism were investigated using human liver microsomes (HLMs), and its cytotoxicity against human hepatoma HepG2 cells was also evaluated. Method Astemizole, a representative CYP2J2 probe substrate, was incubated in HLMs in the presence or absence of acetylshikonin. After incubation, the samples were analyzed by liquid chromatography and triple quadrupole mass spectrometry. The anti-cancer activity of acetylshikonin was evaluated on human hepatocellular carcinoma HepG2 cells. WST-1, cell counting, and colony formation assays were further adopted for the estimation of the growth rate of HepG2 cells treated with acetylshikonin. Results Acetylshikonin inhibited CYP2J2-mediated astemizole O-demethylation activity (Ki = 2.1 µM) in a noncompetitive manner. The noncompetitive inhibitory effect of acetylshikonin on CYP2J2 enzyme was also demonstrated using this 3D structure, which showed different binding location of astemizole and acetylshikonin in CYP2J2 model. It showed cytotoxic effects against human hepatoma HepG2 cells (IC50 = 2 μM). In addition, acetylshikonin treatment inhibited growth of human hepatocellular carcinoma HepG2 cells leading to apoptosis accompanied with p53, bax, and caspase3 activation as well as bcl2 down-regulation. Conclusion Taken together, our present study elucidates acetylshikonin displays the inhibitory effects against CYP2J2 in HLMs and anti-cancer activity in human hepatocellular carcinoma HepG2 cells.

1,25-Dihydroxyvitamin D3 has been known to have the tumor-suppressive activity in various kinds of tumors. However, the exact effect and working mechanism of 1,25-dihydroxyvitamin D3 on the tumor-suppressive activity in human kidney cancer cells remains poorly understood. 1,25-Dihydroxyvitamin D3 has cytotoxicity to ACHN cells and inhibited ACHN cell proliferation compared to the vehicle control. 1,25-Dihydroxyvitamin D3 increased the expression of the cleaved PARP1, active Caspase3, Bax, and Bim but decreased the expression of Bcl2 in ACHN cells. Moreover, 1,25-dihydroxyvitamin D3 down-regulated the phosphorylated Akt and Erk which might lead to apoptosis through activation of FOXO3 in ACHN cells. Transfection of siRNA against FOXO3 attenuated the pro-apoptotic BimEL expression in ACHN cells treated with 1,25-dihydroxyvitamin D3. These results suggest that FOXO3 is involved in the apoptosis induced by 1,25-dihydroxyvitamin D3.

Angiogenesis is closely associated with osteoblast differentiation. Previously, we demonstrated that bone formation can be accelerated by treatment with COMP-Angiopoietin1, a known angiogenic factor. Angiopoietin1 (Ang1) is a specific growth factor that generates stable and mature vasculature through the Tie2 receptor. In this study, we aimed to identify a novel drug that can activate endogenous Ang1 expression as a pharmacological treatment for bone formation. Therefore, Ang1 expression was examined in U2OS osteoblast-like cells treated with 770 drugs from a library of Food and Drug Administration (FDA)-approved drugs by using ELISA for Ang1. l-thyroxine was selected as a novel drug candidate. l-Thyroxine is a synthetic form of the hormone thyroxine, which is used to treat patients with hypothyroidism. Enzyme-linked immunosorbent assays (ELISAs) were performed to test whether Ang1 is induced in a dose-dependent manner in human osteoblast-like cell lines, U2OS and MG63. The effects of l-thyroxine on osteoblast differentiation and mineralization were evaluated by alkaline phosphatase (ALP) activity and Alizarin red s staining. To determine the molecular mechanism, the expression of proteins related to bone formation and differentiation, such as type I collagen (COL1A1), osteocalcin (OC), bone sialoprotein (BSP), distal-less homeobox 5 (Dlx5), Runt-related transcription factor 2 (Runx2), osterix (OSX), and ALP, was tested by Western blotting analysis. Consequently, l-thyroxine induced Ang1 expression in a dose-dependent manner in both U2OS and M63 cells, which was confirmed by ELISA and Western blotting. Also, l-thyroxine activated ALP activity in U2OS and MG63 cells as well as ALP expression. Furthermore, l-thyroxine enhanced the expression of COL1A1, Runx2, OC, BSP, Dlx5, and OSX mRNA and proteins. Taken together, we demonstrated that l-thyroxine increased Ang1 expression and induces bone formation, differentiation, and mineralization in U2OS and MG63 cell lines, which suggests that l-thyroxine could be a potential bone production agent.

Quetiapine, an atypical antipsychotic drug, is used for the treatment of schizophrenia and acute mania. Although a previous report showed that quetiapine blocked hERG potassium current, quetiapine has been considered relatively safe in terms of cardiovascular side effects. In the present study, we used the whole-cell patch-clamp technique to investigate the effect that quetiapine and its major metabolite norquetiapine can exert on human cardiac sodium channels (hNav1.5). The half-maximal inhibitory concentrations of quetiapine and norquetiapine at a holding potential of −90 mV near the resting potential of cardiomyocytes were 30 and 6 μM, respectively. Norquetiapine as well as quetiapine was preferentially bound in the inactivated state of the hNav1.5 channel. Norquetiapine slowed the recovery from inactivation of hNav1.5 and consequently induced strong use-dependent inhibition. Our results indicate that norquetiapine blocks hNav1.5 current in concentration-, state- and use-dependent manners, suggesting that the blockade of hNav1.5 current by norquetiapine may shorten the cardiac action potential duration and reduce the risk of QT interval prolongation induced by the inhibition of hERG potassium currents.

FAM83H is primarily known for its role in amelogenesis; however, recent reports suggest FAM83H might be involved in tumorigenesis. Although the studies of FAM83H in kidney cancer are limited, a search of the public database shows a significant association between FAM83H and pannexin-2 (PANX2) in clear cell renal cell carcinomas (CCRCCs). Therefore, we evaluated the clinicopathological significance of the immunohistochemical expression of FAM83H and PANX2 in 199 CCRCC patients. The expression of FAM83H and PANX2 were significantly associated with each other. In univariate analysis, individual, and co-expression pattern of FAM83H and PANX2 was significantly associated with shorter overall survival (OS) and relapse-free survival (RFS) of CCRCC patients: nuclear expression of FAM83H (OS; P < 0.001, RFS; P < 0.001), cytoplasmic expression of FAM83H (OS; P < 0.001, RFS; P < 0.001), nuclear expression of PANX2 (OS; P < 0.001, RFS; P < 0.001), cytoplasmic expression of PANX2 (OS; P < 0.001, RFS; P < 0.001), co-expression pattern of nuclear FAM83H and nuclear PANX2 (OS; P < 0.001, RFS; P < 0.001). In multivariate analysis, nuclear expression of FAM83H (OS; P < 0.001, RFS; P = 0.003) and the co-expression pattern of nuclear FAM83H and PANX2 (OS; P < 0.001, RFS; P < 0.001) were independent indicators of shorter survival of CCRCC patients. Cytoplasmic expression of FAM83H was associated with shorter RFS (P = 0.030) in multivariate analysis. In Caki-1 and Caki-2 CCRCC cells, knock-down of FAM83H decreased PANX2 expression and cell proliferation, and overexpression of FAM83H increased PANX2 expression and cell proliferation. These results suggest that FAM83H and PANX2 might be involved in the progression of CCRCC in a co-operative manner, and their expression might be used as novel prognostic indicators for CCRCC patients.

Melanogenesis is the process of production of melanin pigments that are responsible for the colors of skin, eye, and hair and provide protection from ultraviolet radiation. However, excessive levels of melanin formation cause hyperpigmentation disorders such as freckles, melasma, and age spots. Liver X receptors (LXR) are nuclear oxysterol receptors belonging to the family of ligand-activated transcription factors and physiological regulators of lipid and cholesterol metabolism. In the skin, activation of LXRs stimulates differentiation of keratinocytes and augments lipid synthesis in sebocytes. However, the function of LXRs in melanogenesis has not been clearly elucidated. In addition, although beauvericin, a well-known mycotoxin primarily isolated from several fungi, has various biological properties, its involvement in melanogenesis has not been reported. Therefore, in this study, we examined the effects of beauvericin on melanogenesis and its molecular mechanisms. Beauvericin decreased melanin content and tyrosinase activity without any cytotoxicity. Beauvericin also reduced protein levels of MITF, tyrosinase, TRP1, and TRP2. In addition, beauvericin suppressed cAMP-PKA-CREB signaling and upregulated expression of LXR-α, resulting in the suppression of p38 MAPK. Our results indicate that beauvericin attenuates melanogenesis by regulating both cAMP/PKA/CREB and LXR-α/p38 MAPK pathways, consequently leading to a reduction of melanin levels.

Urban particulate matter (UPM) exerts negative effects on various human organs. Transient receptor potential vanilloid 1 (TRPV1) is a polymodal sensory transducer that can be activated by multiple noxious stimuli. This study aimed to explore the effects of the UPM 1648a on the expression of TRPV1, and its regulatory mechanisms in HaCaT cells. UPM enhanced TRPV 1 promoter-luciferase reporter activity. UPM also increased expression of the TRPV 1 gene as evidenced by increased mRNA and protein levels of TRPV 1. In addition, elucidation of the underlying mechanism behind the UPM-mediated effects on TRPV 1 expression revealed that UPM can upregulate expression of the TRPV1 gene by activating activator protein-1 (AP-1) and nuclear factor kappa B (NF-&kappa, B). The UPM treatment also altered Ca2+ influx and cell proliferation, as well as production of interleukin-8 (IL-8), tumor necrosis factor-&alpha, (TNF-&alpha, ), and interleukin-1&beta, (IL-1&beta, ). In addition, these UPM-induced effects were attenuated by SB203580 and ammonium pyrrolidinedithiocarbamate (PDTC). However, SP600125 and PD98059 did not alter the UPM-induced effects. Taken together, these findings indicate that UPM upregulates expression of the TRPV 1 gene, which is mediated by the p38 mitogen-activated protein kinase (MAPK) and NF-&kappa, B signaling pathways and suggest that UPM is a potential irritant that can induce skin processes such as aging and inflammatory responses.

Background PARP1 facilitates the recovery of DNA-damaged cells by recruiting DNA damage response molecules such as γH2AX and BRCA1/2, and plays a role in resistance to antitumor therapies. Therefore, PARP inhibition being evaluated as an anti-cancer therapy. However, there are limited studies regrading PARP inhibition in osteosarcoma. Methods We evaluated the expression of DNA damage response molecules in 35 human osteosarcomas and investigated the effects of co-treatment of the PARP inhibitor, olaparib, and doxorubicin in osteosarcoma cells. Results The expression patterns of PARP1, γH2AX, BRCA1, and BRCA2 were significantly associated with shorter survival of osteosarcoma patients. In osteosarcoma cells, knock-down of PARP1 and treatment of olaparib significantly inhibited proliferation of cells and induced apoptosis. Moreover, the anti-tumor effect was more significant with co-treatment of olaparib and doxorubicin in vitro and in vivo. Conclusions This study suggests that combined use of a PARP inhibitor with doxorubicin, a DNA damaging agent, might be effective in the treatment of osteosarcoma patients, especially in the poor-prognostic subgroups of osteosarcoma expressing PARP1, γH2AX, or BRCA1/2.

The outcome of chemotherapy for osteosarcoma have improved during the past decade and more patients have access to combination chemotherapy, but there has been no significant clinical progress in the patient survival rate. Recently, forkhead-box O3 (FOXO3) was identified as a pivotal transcription factor responsible for the transcriptional regulation of genes associated with suppression of cancer. The purpose of the present study was to screen small chemicals activating FOXO3 and elucidate their underlying mechanism. Using a drug discovery platform based on the phosphorylation status of FOXO3 in osteosarcoma cells, mitoxantrone (MTZ), a type of DNA-damaging agent, was selected as a possible FOXO3 activator from the food and drug administration-approved drug library. MTZ treatments significantly inhibited the phosphorylation level of Akt-pS473 and caused nuclear localization of FOXO3 in osteosarcoma cells. MTZ treatment inhibited proliferation in osteosarcoma cells in vitro, whereas silencing FOXO3 potently attenuates MTZ-mediated apoptosis in osteosarcoma cells. Taken together, the results indicated that MTZ induces apoptosis in osteosarcoma cells through an Akt/FOXO3-dependent mechanism.

Indole-3-carbinol (I3C) is a chemical found in many cruciferous vegetables such as cabbage that is known to have anti-cancer effects. However, the effects of I3C on liver cancer have not been well studied. Here, we examined the anti-cancer effects of I3C in human hepatocellular carcinoma Huh-7 cells. We observed that treatment of Huh-7 cells with I3C decreased viability by MTT and colony formation assays. Fluorescence-activated cell sorter (FACS) analysis showed that I3C induced early and late apoptosis in a dose-dependent manner. We also observed that I3C induced DNA fragmentation via terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) assay. Differential protein expression in untreated control and 500 µM I3C-treated Huh-7 cells was accessed by 2-dimensional gel electrophoresis (2-DE). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) identified heat shock protein 27 (Hsp27) as one of the proteins that was upregulated in I3C-treated cells. Western blotting results showed that various apoptotic proteins, such as p53, cleaved caspase-3, -7, -9, and poly ADP-ribose polymerase (PARP) were activated in Huh-7 cells following treatment with I3C. Transfection of Hsp27 siRNA was performed to investigate the effects of Hsp27 in Huh-7 cells. We concluded that I3C induced Hsp27 overexpression and subsequent apoptosis in Huh-7 cells.

This study was focused on investigating the anticancer potential of indole-3-carbinol (I3C) against osteosarcoma MG-63 and U2OS cells. A wound healing assay indicated that IC3 inhibited migration of MG-63 and U2OS cells. MTT, WST-1, and colony formation assays revealed that treatment of MG-63 and U2OS cells with I3C decreased cell viability. Fluorescence-activated cell sorting (FACS) analysis showed that I3C induced apoptosis in a dose- and time-dependent manner in MG-63 and U2OS cells. Moreover, via terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP-biotin nick-end labeling (TUNEL) assay, we detected that I3C induced DNA fragmentation. Western blotting demonstrated that activated forms of caspase-3, caspase-7, and caspase-9, as well as poly (ADP-ribose) polymerase (PARP) were increased in MG-63 and U2OS cells, following treatment with I3C. Furthermore, protein expression levels of FOXO3, Bax, and Bim extra-large form were increased while those of Akt, JNK, p38, phosphorylated ERK, and Bcl-xL were decreased by I3C treatment in MG-63 and U2OS cells. Thus, the study indicates that I3C may induce apoptosis in human osteosarcoma MG-63 and U2OS cells via the activation of apoptotic signaling pathways by FOXO3.

The flavonoid myricetin (MYR) is derived from vegetables and fruits. It has been shown to exert anti-cancer effects in various cell lines; however, the exact mechanism underlying these effects is yet to be elucidated. In this study, we evaluated the anti-cancer effects induced by MYR treatment in colon cancer HCT-15 cells. To detect cell proliferation, we conducted MTT assay and real time-cell electronic sensing (RT-CES). We next performed comet assay and Annexin V and PI staining to detect cellular apoptotic features. After that, we conducted two-dimensional electrophoresis (2-DE) analysis to identify apoptotic proteins. The results of this analysis revealed that eight spots were differentially expressed. Among the spots, we selected nucleoside diphosphate kinase (NDPK) for further investigation, as it has been shown to regulate cancer cell apoptosis and metastasis. After that, we conducted realtime-PCR and western blot to detect the expression of NDPK mRNA and protein and wound-healing assay to detect cell migration and invasion. Finally, we performed NDPK siRNA transfection study and the results showed that NDPK knockdown inhibited apoptosis. Based on these collective results, we suggest that MYR induces apoptosis in human colon cancer HCT-15 cells selectively by increasing the expression of NDPK and other caspase-regulated apoptosis proteins.

The stemness of stem cells is negatively affected by ultraviolet A (UVA) irradiation. This study was performed to examine the effects of arctigenin on UVA-irradiation-induced damage to the stemness of human mesenchymal stem cells (hMSCs) derived from adipose tissue. The mechanisms of action of arctigenin were also investigated. A BrdU-incorporation assay demonstrated that arctigenin attenuated the UVA-induced reduction of the cellular proliferative potential. Arctigenin also increased the UVA-induced reduction in stemness of hMSCs by upregulating stemness-related genes such as SOX2, OCT4, and NANOG. In addition, the UVA-induced reduction in the mRNA expression level of hypoxia-inducible factor (HIF)-1α was significantly recovered by arctigenin. The antagonizing effect of arctigenin on UVA irradiation was mediated by reduced PGE2 production through the inhibition of MAPKs (p42/44 MAPK, p38 MAPK, and JNK) and NF-κB. Overall, these findings suggest that arctigenin can ameliorate the reduced stemness of hMSCs induced by UVA irradiation. The effects of arctigenin are mediated by PGE2-cAMP signaling-dependent upregulation of HIF-1α. Therefore, arctigenin could be used as an antagonist to attenuate the effects of UVA irradiation.

Ultraviolet A (UVA) irradiation exerts negative effects on stemness and differentiation potential of stem cells. This study aimed to explore the effect of sinapic acid on UVA-irradiation-induced damages to stemness and differentiation potential of human-adipose-tissue-derived mesenchymal stem cells (hAMSCs) and its UVA-antagonist mechanisms. Sinapic acid attenuated UVA-induced reduction in the proliferative potential and stemness by upregulating OCT4, SOX2, and NANOG. In addition, sinapic acid significantly recovered UVA-induced reduction in expression level of hypoxia-inducible factor (HIF)-1α. The antagonist effect of sinapic acid against stemness damage was mediated by reduceing PGE2 production through inhibition of p38 MAPK and NF-κB. Moreover, sinapic acid attenuated UVA-induced reduction in differentiation potential by downregulating the expression of macrophage migration inhibitory factor (MIF) and Kruppel-like factor (KLF) 2 gene while activating AMP-activated protein kinase (AMPK). UVA-induced inhibition of adipogenic differentiation was mediated by reducing MIF production through suppression of NF-κB. Taken together, these findings suggest that sinapic acid may ameliorate UVA-irradiation-induced reduced stemness and differentiation potential of hAMSCs. Therefore, sinapic acid might have potential as an antagonist agent to attenuate damages caused by UVA.

Previously, we reported that auranofin induces apoptosis in SKOV3 cells via regulation of the IKKβ/FOXO3 pathway. In the present study, we reveal that the anticancer activity of auranofin in SKOV3 cells could be enhanced by the attenuation of MUC4 through the regulation of the Her2/Akt/FOXO3 pathway. Compared to the control-siRNA, siRNA transfection against MUC4 into SKOV3 cells accelerated the protein degradation of Her2. Under the same conditions, the expression level of phosphorylated Akt was also downregulated leading to an increase of FOXO3 in the nucleus. Notably, auranofin treatment in SKOV3 cells also resulted in the downregulation of the expression levels of both Her2 and phosphorylated Akt. Thus, Her2 was identified as the common molecular target protein by siRNA transfection against MUC4. Western blot analysis of total and nuclear fraction lysates from SKOV3 cells revealed that attenuation of MUC4 combined with auranofin treatment in SKOV3 cells synergistically activated FOXO3 translocation from the cytoplasm to the nucleus through the regulation of the Her2/Akt/FOXO3 pathway. Attenuation of MUC4 by siRNA transfection potentiated the antitumor effect of auranofin which was examined by performing in vitro assays such as WST-1, cell counting, colony formation, TUNEL and Annexin V staining. In addition, western blot analysis of the apoptosis‑related proteins such as PARP1, caspase-3, Bim extra large (EL), Bax and Bcl2 revealed that the attenuation of MUC4 by siRNA transfection potentiates the pro-apoptotic activity of auranofin in SKOV3 cells. Collectively, auranofin could regulate the Her2/Akt/FOXO3 signaling pathway in SKOV3 cells and be used as a potential antitumor agent considering the expression of MUC4 in ovarian cancer patients.

Tomentosin is a natural sesquiterpene lactone extracted from various plants and is widely used as a medicine because it exhibits essential therapeutic properties. In this study, we investigated the anti-carcinogenic effects of tomentosin in human osteosarcoma MG-63 cells by performing cell migration/viability/proliferation, apoptosis, and reactive oxygen species (ROS) analysis assays. MG-63 cells were treated with various doses of tomentosin. After treatment with tomentosin, MG-63 cells were analyzed using the MTT assay, colony formation assay, cell counting assay, wound healing assay, Boyden chamber assay, zymography assay, cell cycle analysis, FITC Annexin V apoptosis assay, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, western blot analysis, and ROS detection analysis. Our results indicated that tomentosin decreased cell viability and migration ability in MG-63 cells. Moreover, tomentosin induced apoptosis, cell cycle arrest, DNA damage, and ROS production in MG-63 cells. Furthermore, tomentosin-induced intracellular ROS decreased cell viability and induced apoptosis, cell cycle arrest, and DNA damage in MG-63 cells. Taken together, our results suggested that tomentosin exerted anti-carcinogenic effects in MG-63 cells by induction of intracellular ROS.

Recently, the roles of FAM83H in tumorigenesis have been interested and increased expression of FAM83H and MYC in hepatocellular carcinoma (HCC) have been reported. Therefore, we investigated the expression and role of FAM83H in 163 human HCCs and further investigated the relationship between FAM83H and oncogene MYC. The expression of FAM83H is elevated in liver cancer cells, and nuclear expression of FAM83H predicted shorter survival of HCC patients. In HLE and HepG2 HCC cells, knock-down of FAM83H inhibited proliferation and invasive activity of HCC cells. FAM83H induced expression of cyclin-D1, cyclin-E1, snail and MMP2 and inhibited the expression of P53 and P27. In hepatic tumor cells derived from Tet-O-MYC mice, the expression of mRNA and protein of FAM83H were dependent on MYC expression. Moreover, a chromatin immunoprecipitation assay demonstrated that MYC binds to the promotor of FAM83H and that MYC promotes the transcription of FAM83H, which was supported by the results of a dual-luciferase reporter assay. In conclusion, we present an oncogenic role of FAM83H in liver cancer, which is closely associated with the oncogene MYC. In addition, our results suggest FAM83H expression as a poor prognostic indicator of HCC patients.

Melanogenesis refers to synthesis of the skin pigment melanin, which plays a critical role in the protection of skin against ultraviolet irradiation and oxidative stressors. We investigated the effects of afzelin on melanogenesis and its mechanisms of action in human epidermal melanocytes. In this study, we found that afzelin increased both melanin content and tyrosinase activity in a concentration-dependent manner. While the mRNA levels of microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related protein (TRP)-1 increased following afzelin treatment, the mRNA levels of TRP-2 were not affected by afzelin. Likewise, afzelin increased the protein levels of MITF, TRP-1, and tyrosinase but not TRP-2. Mechanistically, we found that afzelin regulated melanogenesis by upregulating MITF through phosphorylation of p38 mitogen-activated protein kinase (MAPK), independent of cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling. Taken together, these findings indicate that the promotion of melanogenesis by afzelin occurs through increased MITF gene expression, which is mediated by activation of p38 MAPK, and suggest that afzelin may be useful as a protective agent against ultraviolet irradiation.

Recently, the roles of sirtuins (SIRTs) in tumorigenesis have been of interest to oncologists, and protein kinase CK2 α1 (CSNK2A1) has been shown to be involved in tumorigenesis by phosphorylating various proteins, including SIRT1. Therefore, we evaluated the roles of CSNK2A1, SIRT6, and phosphorylated SIRT6 and their relationships in breast carcinoma. Nuclear expression of CSNK2A1 and SIRT6 predicted shorter overall survival and relapse-free survival by multivariate analysis. Inhibition of CSNK2A1 decreased the proliferative and invasive activity of cancer cells. In addition, CSNK2A1 was bound to SIRT6 and phosphorylated SIRT6; evidence for this is provided from immunofluorescence staining, co-immunoprecipitation of CSNK2A1 and SIRT6, a glutathione S -transferase pull-down assay, an in vitro kinase assay, and transfection of mutant CSNK2A1 . Knockdown of SIRT6 decreased the proliferation and invasiveness of cancer cells. Overexpression of SIRT6 increased proliferation, but mutation at the Ser338 phosphorylation site of SIRT6 inhibited the proliferation of MCF7 cells. Moreover, both knockdown of SIRT6 and a mutation at the phosphorylation site of SIRT6 decreased expression of matrix metallopeptidase 9, β-catenin, cyclin D1, and NF-κB. Especially, SIRT6 expression was associated with the nuclear localization of β-catenin. This study demonstrates that CSNK2A1 and SIRT6 are indicators of poor prognosis for breast carcinomas and that CSNK2A1-mediated phosphorylation of SIRT6 might be involved in the progression of breast carcinoma.

// See-Hyoung Park 1, 2, * , Young Min Chung 1, * , Jessica Ma 1 , Qin Yang 3 , Jonathan S. Berek 1 , Mickey C-T. Hu 1 1 Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA 94305, USA 2 Current address: Department of Biological and Chemical Engineering, Hongik University, Sejong, 339-701, Korea 3 Cancer Biology Division, Department of Radiation Oncology, Washington University School of Medicine, Saint Louis, MO 63108, USA * These authors contributed equally to this work Correspondence to: Mickey C-T. Hu, email: mhu1@stanford.edu Keywords: FOXO3, breast cancer, bepridil, trifluoperazine, dopamine receptor Received: January 18, 2016 Accepted: May 12, 2016 Published: June 07, 2016 ABSTRACT Triple-negative breast cancer (TNBC) is the most lethal form of breast cancer. Lacking effective therapeutic options hinders treatment of TNBC. Here, we show that bepridil (BPD) and trifluoperazine (TFP), which are FDA-approved drugs for treatment of schizophrenia and angina respectively, inhibit Akt-pS473 phosphorylation and promote FOXO3 nuclear localization and activation in TNBC cells. BPD and TFP inhibit survival and proliferation in TNBC cells and suppress the growth of TNBC tumors, whereas silencing FOXO3 reduces the BPD- and TFP-mediated suppression of survival in TNBC cells. While BPD and TFP decrease the expression of oncogenic c-Myc, KLF5, and dopamine receptor DRD2 in TNBC cells, silencing FOXO3 diminishes BPD- and TFP-mediated repression of the expression of these proteins in TNBC cells. Since c-Myc, KLF5, and DRD2 have been suggested to increase cancer stem cell-like populations in various tumors, reducing these proteins in response to BPD and TFP suggests a novel FOXO3-dependent mechanism underlying BPD- and TFP-induced apoptosis in TNBC cells.

Ultraviolet A (UVA) irradiation induces various changes in cell biology. The objective of this study was to determine the effect of vanillin on UVA irradiation-induced damages in the stemness properties of human adipose tissue-derived mesenchymal stem cells (hAMSCs). UVA-antagonizing mechanisms of vanillin were also examined. The results revealed that vanillin attenuated UVA-induced reduction of the proliferative potential and stemness of hAMSCs evidenced by increased proliferative activity in BrdU incorporation assay and upregulation of stemness-related genes (OCT4, NANOG and SOX2) in response to vanillin treatment. UVA-induced reduction in mRNA level of hypoxia-inducible factor (HIF)-1α was significantly recovered by vanillin. In addition, the antagonizing effect of vanillin on UVA was found to be mediated by reduced production of PGE2 through inhibiting JNK and p38 MAPK. Taken together, these findings showed that vanillin could improve the reduced stemness of hAMSCs induced by UVA. The effect of vanillin is mediated by upregulating HIF-1α via inhibiting PGE2-cAMP signaling. Therefore, vanillin might be used as an antagonizing agent to mitigate the effects of UVA.

DNA damage response (DDR) molecules are protective against genotoxic stresses. DDR molecules are also involved in the survival of cancer cells in patients undergoing anti-cancer therapies. Therefore, DDR molecules are potential markers of cancer progression in addition to being potential therapeutic targets. In this study, we evaluated the immunohistochemical expression of PARP1, gamma H2AX, BRCA1, and BRCA2 and their prognostic significance in 112 cases of soft tissue sarcoma (STS). The expression of PARP1, gamma H2AX, BRCA1, and BRCA2 were significantly associated with each other and were associated with higher tumor stage and presence of distant metastasis. The expression of PARP1,gamma H2AX, and BRCA2 were significantly associated with shorter disease-specific survival (DSS) and event-free survival (EFS) by univariate analysis. BRCA1 expression was associated with shorter DSS. Multivariate analysis revealed the expression of PARP1 and gamma H2AX to be independent indicators of poor prognosis of DSS and EFS. BRCA2 expression was an independent indicator of poor prognosis of DSS. In addition, the combined expressional patterns of PARP1, gamma H2AX, BRCA1, and BRCA2 (CSddrm) were independent prognostic predictors of DSS (P < 0.001) and EFS (P = 0.016). The ten-year DSS rate of the CSddrm-low, CSddrm-intermediate, and CSddrm-high subgroups were 81%, 26%, and 0%, respectively. In conclusion, this study demonstrates that the individual and combined expression patterns of the DDR molecules PARP1, gamma H2AX, BRCA1, and BRCA2 could be predictive of the prognosis of STS patients and suggests that controlling the activity of these DDR molecules could be employed in new therapeutic stratagems for the treatment of STS.

Gas (SF6)-filled microbubbles (MBs) were prepared by emulsion and solvent-evaporation method. The prepared MBs were further conjugated with doxorubicin (Dox)-loaded nano-sized liposome and peptide ligands to interleukin-4 receptor (IL4R) for targeting brain tumor cells. The final MB-liposome (Dox)-IL4R targeting peptide ligand [MB-Lipo (Dox)-IL4RTP] had a spherical structure with the mean size of 1,500 nm. The MB-Lipo (Dox)‑IL4RTP exhibited cellular uptake in U87MG brain tumor cells (a brain tumor cell line expressing strongly IL4R) with frequency ultrasound energy suggesting that MB-Lipo (Dox)‑IL4RTP provided effective targeting ability for brain tumor cells. In addition, WST-1 assay results showed that MB-Lipo (Dox)‑IL4RTP inhibited the proliferation of U87MG cells IL4R‑dependently. This was confirmed by western blotting of γH2AX, phospho (Ser15)-p53, p53 and p21 which are signal transduction proteins involved in DNA damage response and cell cycle arrest. Taken together, these results indicate that MB-Lipo (Dox)-IL4RTP represents a promising ultrasonic contrast agent for tumor-targeting ultrasonic imaging.